AB0019 RITUXIMAB TREATMENT DOES NOT ALTER EXPRESSION OF CO-STIMULATION MARKER CD19 ON B CELLS IN SYSTEMIC SCLEROSIS PATIENTS
Authors: B. Dreo, B. Prietl, S. Kofler, V. Pfeifer, H. Sourij, F. Moazedi-Fürst, S. Kielhauser, M. D’Orazio, S. Zenz, J. Thiel, M. Stradner, H. P. Brezinsek
Journal: Annals of the Rheumatic Diseases (2022)

Background CD19 is a membrane glycoprotein interacting with different surface molecules like the B cell receptor (BCR) and is crucial for antigen-independent development as well as immunoglobulin-induced activation of B cells.[1] Alterations in this signalling pathway can incline autoantibody production and systemic autoimmunity in humans. Rituximab (RTX), a CD20 antagonist appears to be an effective candidate in the treatment of different autoimmune diseases that are partly driven by autoreactive B cells, such as systemic sclerosis (SSc).[2] It has been speculated that RTX might work not only by depleting B cells but also to down regulate activation markers, such as CD19. Objectives In-depth analysis of CD19 abundancy and activation on B cells in SSc patients with and without RTX treatment Methods Peripheral blood samples from 41 patients suffering from SSc (median ± standard deviation SD, age: 54.3 ± 10.6 years, female ratio: 0.8) and 45 age- and sex-matched healthy controls (HC) (age: 51.0 ± 13.9 years, female ratio: 0.8) were drawn and PBMCs were isolated on-site. We performed flow cytometry analysis on a standardized BD LSRFortessa platform to identify B cell (CD19+CD20+) subpopulations. The geometric mean fluorescence intensity (gMFI) for CD19 in all B cell subtypes was extracted from the data set and used for further statistical analysis. Additionally, a quantitative flow cytometric bead-based assay (QuantiBRITE PE kit from Becton Dickinson) was used for the estimation of CD19 antibodies bound per cell. Results 3 out of 41 SSc patients were in high disease activity at the time of blood drawal. 23 SSc patients were under RTX therapy of whom 5 patients still displayed measurable B cells frequencies. Naïve B cells made up the most abundant B cell population in SSc patients. Thus, the frequency of IgM+/IgD+/CD27- B cells was 67.9% ±13.2 (mean ±SD), followed by class-switched memory B cells (IgM-/IgD-/CD27+, 10.5 ± 4.9), non-switched memory B cells (IgM+/IgD+/CD27+, 4.0 ± 3.6) and plasmablasts (0.3 ± 4.4). Pairwise Wilcoxon Tests (Bonferroni-corrected for multiple testing) showed significant differences (p &lt; 0.001) between frequencies of naïve B cells and all other cell types. In contrast, naïve B cells displayed the second lowest CD19 gMFI levels (7601.0 ± 1912.0) in the dataset. Non-switched memory B cells in SSc patients showed the highest CD19 gMFI (10620.0 ± 15689.8), followed by class-switched (9388 ± 3048.6). As expected, Plasmablasts displayed the lowest CD19 gMFI levels (4799.0 ± 4185.7). The decrease in CD19 gMFI was again highly significant. This trajectory in decreasing CD19 gMFI was found in both HCs and SSc patients. We saw a significant reduction in percentages of non-switch B cells and class-switched B cells in SSc patients compared to HCs (4.0 ± 3.6 vs 6.5 ± 4.2, p = 0.029, 10.5 ± 4.9 vs 13.2 ± 7.2 p = 0.04) but an increase in CD19 gMFI in non-switched B cells (HC: 9204.5 ± 2116.8, p = 0.05). Interestingly, SSc patients under RTX treatment had significantly lower class-switched memory B cell frequencies compared to HCs (6.4 ± 4.2 vs. 13.2 ± 7.2, p = 0.015). However, RTX did not affect CD19 gMFI or bound CD19 in SSc. Conclusion RTX treatment in SSc is not associated with downregulation of the co-stimulation marker CD19. Thus, the main effect of this drug is the reduction of B cells, especially class-swtched memory B cells that might have a high capacity to activate other cells involved in the pathogenesis of SSc. References [1]M. Wang et al. , “Identification and Validation of Predictive Biomarkers to CD19- and BCMA-Specific CAR T-Cell Responses in CAR T-Cell Precursors,” Blood , vol. 134, no. Supplement_1, pp. 622–622, Nov. 2019, doi: 10.1182/blood-2019-122513. [2]S. Ebata et al. , “Safety and efficacy of rituximab in systemic sclerosis (DESIRES): a double-blind, investigator-initiated, randomised, placebo-controlled trial,” Lancet Rheumatol. , 2021. Acknowledgements This work was funded by a grant from JDRF, LRA and NMSS (grant key: 2-SRA-2021-1043-S-B) and the Austrian Federal Government within the COMET K1 Centre Program, Land Steiermark and Land Wien. Disclosure of Interests Barbara Dreo: None declared, Barbara Prietl: None declared, Selina Kofler: None declared, Verena Pfeifer: None declared, Harald Sourij: None declared, Florentine Moazedi-Fürst: None declared, Sonja Kielhauser: None declared, Monica D’Orazio: None declared, Sabine Zenz: None declared, Jens Thiel Speakers bureau: Novartis, GSK, Vifor, BMS, Consultant of: Novartis, GSK, Vifor, Grant/research support from: BMS, Martin Stradner: None declared, Hans-Peter Brezinsek: None declared